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The aim of this study was to investigate the temporal involvement of different proteolytic systems and muscle proteome changes during experimental disuse atrophy (up to 1 week hindlimb suspension, HS) in murine gastrocnemius muscle. The results showed that proteolysis, cytoprotection mechanisms and signs of cellular infiltration occurred very early. After 1 day of HS, signals of lysosomal activation, rather than programmed cell death (apoptosis), seem to trigger protein breakdown in the whole skeletal muscle. Moreover, the ubiquitin-proteasome pathway remained elevated later whereas all other proteolytic parameters returned to control values when atrophy was fully established. Using proteomics, evidence is provided for metabolic alterations toward glycolysis and for cytoskeleton remodelling suggestive of reduced capacity for force generation. Overall, our data highlight an early and coordinated time-dependent activation of proteolysis, which explains the global proteome alterations observed in gastrocnemius under atrophic conditions.

The purpose of the present study was to investigate the mechanisms of cell death (apoptosis vs. necrosis) during muscle atrophy induced by 1 week of hindlimb suspension. Biochemical and morphological parameters were examined in murine soleus and gastrocnemius muscles. A total of 70 male Charles River CD1 mice were randomly assigned to seven groups (n = 10/group): Cont (loading control conditions) and 6HS, 12HS, 24HS, 48HS, 72HS and 1wkHS with respect to the period of hindlimb suspension (HS). Compared to the Cont, skeletal muscle atrophy was confirmed by a significant decrease of 44 and of 17% in fiber cross-sectional areas in the gastrocnemius and soleus, respectively. A significant increase in caspase-3 activity was noticed in 6HS (196%, P < 0.05) and in 12HS (201%, P < 0.05), as well as the amount of cytosolic mono- and oligonucleosomes at 12HS (142%, P < 0.05) and 24HS (203%, P < 0.05) in the gastrocnemius and soleus, respectively. The profile of necrotic markers showed a peak of myeloperoxidase activity at 24HS (170%, P < 0.05) and at 72HS (114%, P < 0.05) in the gastrocnemius and soleus, respectively. The analysis of N-acetylglucosaminidase activity evidenced more increment in the soleus at 72HS (60%, P < 0.05). The analysis of the basal values of these parameters suggested that apoptosis prevailed in the slow-twitch muscle analyzed, whereas lysosomic activity seemed to be more pronounced in the gastrocnemius. The morphological data supported the biochemical results pointing towards a shift from apoptosis to necrosis, which seems to corroborate the aponecrosis theory.

The effects of a high-altitude exposure were studied in six mountaineers who spent 3 weeks at an altitude range between 5,250 and 7,161 m after 1 week in an acclimatization trek (2,800-5,250 m). Blood drawn from the antecubital vein was collected at sea level 1 day before and 1 day after the expedition to analyse some haematological variables [haemoglobin (Hb), haematocrit (Htc) and red blood cell (RBC) count], erythrocyte antioxidant enzyme activity [superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (Gr)] and membrane fatty acid profile [mono-unsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), saturated fatty acids (SFA), trans fatty acids (TRANS)]. Moreover, total antioxidant status (TAS), thiobarbituric acid reactive substances (TBARS), thiol protein groups (SH), SOD, GPx and Gr were measured in plasma. High-altitude exposure induced polycythaemia, with significant increases in RBC count (5.26%), Hb concentration (4.83%) and Htc (6.26%). Furthermore, a significant increase in plasma TBARS, SOD and Gr was observed after the expedition, whereas SH, TAS and GPx decreased. Erythrocyte glutathione-cycle-related antioxidant enzyme activity was upregulated, whereas SOD activity was maintained after the expedition. In addition, despite the unchanged (MUFA+PUFA)/SFA ratio, the membrane erythrocyte fatty acid content showed a significant increase in PUFAs and a decrease in TRANS, suggesting enhanced membrane fluidity. In conclusion, it seems that high-altitude exposure, besides quantitative variations in RBC expression, induced plasma oxidative stress and damage, and significant changes in erythrocyte components, namely in antioxidant enzyme activity and membrane fatty acid profile that might modify RBC functionality.

This study intended to analyze: (1) the effects of acute and severe hypoxia exposure on skeletal muscle oxidative stress and oxidative damage markers; (2) the protective role of the antioxidant glutathione against oxidative damage; and (3) the expression of heat shock protein 70 kDa (HSP70) induced by this hypoxic insult. Forty mice were divided into four groups: control + placebo (C+P), hypoxia + placebo (H+P), control + l-buthionine-[ S, R]-sulfoximine (BSO, a GSH-depleting compound) (C+BSO) and hypoxia + BSO (H+BSO). Hypoxia groups were continuously exposed for 24 h to a hypobaric hypoxic environment equivalent to an altitude of 7000 m and sacrificed immediately after. Control groups were maintained at sea level during the experimental protocol. Analyzed biochemical parameters were: reduced (GSH) and oxidized (GSSG) glutathione, thiobarbituric acid reactive substances (TBARS), sulfhydryl protein groups (SH), N-acetyl-beta- d-glucosaminidase (NAG) and HSP70 protein. Hypoxia (H+P) per se, compared to C+P, induced a significant increase in %GSSG (5.68 vs. 1.14%), TBARS (436.7 vs. 227.9 nM), NAG (4.49 vs. 3.35 U/mg) and HSP70 (178.7 vs. 100%). Compared with H+P, H+BSO showed a significant decrease in total glutathione (19.30 vs. 6.13 nmol/mg) and an additional increase in %GSSG (5.68 vs. 11.33%) and in HSP70 expression (178.7 vs. 202.2%). However, no further oxidative damage was observed in H+BSO. These data suggest that acute hypoxia per se might enhance oxidative stress; however, the glutathione system seems to have a modest role in skeletal muscle protection against hypoxia-induced oxidative stress. Moreover, hypoxia and BSO treatment is a sufficient stimulus to promote HSP70 overexpression.

The comprehension of the cellular mechanisms underlying skeletal muscle atrophy has been the aim of several experimental studies. However, the majority of them focused on alterations of the myocytes induced by different experimental conditions yet disregarding the contribution of other cells such as endothelial cells and fibroblasts. In this sense, 70 Charles River CD1 male mice were randomly assigned to seven groups (n = 10 per group): control and 6, 12, 24, 48, 72 h and 1 week with respect to the period of hindlimb suspension. Forty-eight hours before sacrifice, the animals were injected with bromodeoxyuridine (BrdU) in order to identify proliferating cells. Immunohistochemistry and south-western blotting techniques were used to evaluate across the whole gastrocnemius muscle BrdU incorporation into the different proliferating cells. The contribution of the apoptotic response was also measured in order to ascertain whether the balance between cell survival and death was preserved. The results observed during 1 week of unloading-induced atrophy evidenced an intense peak of proliferating activity only after 6 h, mainly due to the duplication of satellite cells. Consequently to this unexpected activation of satellite cells, the addition of nuclei to the fibre syncytium was recognized at 12 h of unloading. After 48 h of weightlessness, the proliferating activity observed was largely due to an interstitial fibrosis. According to the apoptotic index profile observed during the analysed unloading period, this general proliferative activity was balanced by apoptosis, which strongly suggests the existence of a regulatory feedback response between anabolic and catabolic events in unloading-induced skeletal muscle atrophy.